Summary of Work: Improved understanding of the structure of HIV proteins can be useful in unraveling the function of the proteins and in understanding the mechanisms of the function of these proteins. This structural knowledge can also be useful in designing novel anti-HIV agents. X-Ray crystallography is the most accurate technique for determination of protein structures. Major drawbacks of the technique, however, are that it can be very time consuming and is dependent on the availability of protein crystals. Molecular modeling programs, on the other hand, can provide insights into the protein structure based on, amongst other things, homology with proteins whose structures are known. Although this approach is attractive, the results have often been less reliable than desired because insufficient information about the protein is available or the degree of homology with proteins of known structure is less than needed for the development of an accurate model. If information such as which amino acid residues are on the surface of the protein is available, however, the quality of the computer generated model can be significantly increased. This project is designed to probe the primary and tertiary structures of HIV proteins using a combination of chemical modifications, enzymatic degradations and mass spectrometric identification. In using commercially available sources of recombinant HIV proteins, we rapidly became aware of discrepancies between the catalog structures and the actual molecular weights of the proteins. We have developed a technique, direct analysis of affinity-bound analytes, and applied it to recombinant His-tagged proteins that are affinity bound to immobilized metal ion affinity columns to determine the actual sequence of two of these HIV proteins, rp24 and rvif. We are continuing to probe the tertiary structure of recombinant HIV. Although portions of the molecule have been crystallized and the structure solved, the manner in which the two domains are configured together is not known. We are proceeding with acetylation of lysine residues on the intact native protein. Relative reactivities of five lysines have been determined, (positions 70, 131, 140, 170 and 158) while the relative reactivities of six other lysines have not been determined as yet. At this point the relative reactivities do not correlate exactly with the solution structures of the constituent domains. This may be due to the microenvironment around the lysine residues or may be due to the manner in which the two domains are connected in the intact molecule. With the present MS/MS capabilities, the acetylation reactions will be repeated and the reactivities of all lysines will be elucidated.